The present invention relates to novel CDC2 like protein kinases (CLK protein kinases). These protein kinases phosphorylate proteins rich in serine and arginine.
The following description of the background of the invention is provided to aid in understanding the invention, but is not admitted to be or describe prior art to the invention.
Cellular signal transduction is a fundamental mechanism whereby extracellular stimuli are relayed to the interior of cells and subsequently regulate diverse cellular processes. One of the key biochemical mechanisms of signal transduction involves the reversible phosphorylation of proteins. Phosphorylation of polypeptides regulates the activity of mature proteins by altering their structure and function. Phosphate most often resides on the hydroxyl moiety (xe2x80x94OH) of serine, threonine, or tyrosine amino acids in proteins. Enzymes that mediate phosphorylation of cellular effectors fall into two classes. While protein phosphatases hydrolyze phosphate moieties from phosphoryl protein substrates, protein kinases transfer a phosphate moiety from adenosine triphosphate to protein substrates. The converse functions of protein kinases and protein phosphatases balance and regulate the flow of signals in signal transduction processes.
Protein kinases and protein phosphatases are typically divided into two groups: receptor and non-receptor type proteins. Receptor protein kinases are comprised of an extracellular domain, a membrane spanning region, and a catalytic domain.
Protein kinases and protein phosphatases are divided further into three classes based upon the amino acids they act upon. Some catalyze the addition or hydrolysis of phosphate on serine or threonine only, some catalyze the addition or hydrolysis of phosphate on tyrosine only, and some catalyze the addition or hydrolysis of phosphate on serine, threonine, and tyrosine.
Membrane association is an important feature of signal transduction. Protein kinases propagate extracellular signals to the inside of the cell by attracting other signaling molecules to the membrane. Schlessinger and Ullrich, 1992, Neuron 9:383-391. For instance, many receptor protein kinases bind an extracellular ligand, dimerize, and cross phosphorylate one another. These phosphate moieties subsequently attract other proteins necessary for propagating the signal within the cell. The molecules that signal downstream of the receptor protein kinases are often nonreceptor protein kinases which propagate and amplify the extracellular signal.
A class of non-receptor protein kinases are implicated in regulating RNA splicing. Fu, 1995 RNA 1:663-680; Staknis and Reed, 1994, Mol. Cell. Biol. 14:7670-7682. These protein kinases phosphorylate polypeptides rich in serine and arginine (SR proteins). SR proteins are characterized as containing at least one amino-terminal RNA recognition motif and a basic carboxyterminal domain rich in serine and arginine residues, often arranged in tandem repeats. Zahler et al., 1992, Genes Dev 6:837-847. Experimental evidence supports the idea that the SR domain is involved in proteinxe2x80x94protein interactions (Kohtz et al., 1994, Nature 368:119-124) as well as protein-RNA interactions (Harada et al., 1996, Nature 380:175-179), and may contribute to a localization signal directing proteins to nuclear speckles. Hedley et al., 1995, Proc. Natl. Acad. Sci. USA 92:11524-11528.
A recent report demonstrated mCLK1, a CDC2 like kinase, interacts with ASF/SF2, SRp20 and hnRNP proteins in a yeast two hybrid system. Because hnRNP-K binds to the protooncogene p95vav, mCLK1 could be implicated in transmitting signals that regulate the expression of the protooncogenes myc and fos in hematopoietic cells. Furthermore, it was demonstrated, that mCLK1 could phosphorylate ASF/SF2 in vitro, suggesting, that SR containing proteins are the natural substrates of mCLK1. Colwill et al., 1996, EMBO J. 15:265-275.
mCLK1 is a dual specificity protein kinase originally isolated in mouse expression libraries (Ben-David et al., 1991, EMBO J. 10:317-325; Howell et al., 1991, Mol. Cell. Biol. 11:568-572) and human (hCLK1, hCLK2, hCLK3), plant (AFC1, AFC2, AFC3) and fly (DOA) CLK protein kinases have since been identified. Johnson and Smith, 1991, J. Biol. Chem. 266:3402-3407; Hanes et al., 1994, J. Mol. Biol. 244:665-672; Bender and Fink, 1994, Proc. Natl. Acad. Sci. USA 91:12105-12109; Yun et al., 1994, Genes. Dev. 8:1160-1173. The amino terminal domain of these proteins is rich in serine and arginine, whereas the catalytic domain can be most similar to CDC2, a serine/threonine protein kinase. Ben-David et al., 1991, EMBO J. 10:317-325.
Both mCLK1 and the Drosophila homologue, DOA, regulate RNA splicing events. Each of these have two alternatively spliced products coding for either the full-length catalytically active protein or a truncated protein lacking the catalytic domain. Yun et al., 1994, Genes. Dev. 8:1160-1173; Duncan et al., 1995, J. Biol. Chem. 270:21524-21531. Identical splice forms were also found in human CLK protein kinases. Hanes et al., 1994, J. Mol. Biol. 244:665-672. The ratio of these splice products appears to be developmentally regulated in Drosophila (Yun et al., 1994, Genes. Dev. 8:1160-1173), and in a tissue and cell type specific manner in mammals. Hanes et al., 1994, J. Mol. Biol. 244:665-672; Duncan et al., 1995, J. Biol. Chem. 270:21524-21531. In addition, the expression of several other, larger transcripts, are observed to be differentially regulated and are shown to represent partially spliced products. Duncan et al., 1995, J. Biol. Chem. 270:21524-21531.
The present invention is based in part upon the isolation and characterization of nucleic acid molecules encoding CLK serine/threonine kinases designated mCLK2, mCLK3, and mCLK4. CLK serine/threonine kinases regulate RNA splicing in cells and some are highly expressed in cancer cells as well as testis. Various mCLK2, mCLK3, and mCLK4 related molecules and compounds can now be designed as treatments of cancers or as contraceptives to reproduction in male organisms.
The present invention is based in part upon nucleic acid molecules encoding novel mCLK2, mCLK3, and mCLK4 polypeptides, nucleic acid molecules encoding portions of their amino acid sequences, nucleic acid vectors harboring such nucleic acid molecules, cells containing such nucleic acid vectors, purified polypeptides encoded by such nucleic acid molecules, and antibodies to such polypeptides, and methods of identifying compounds that bind mCLK2, mCLK3, and mCLK4 or abrogate their interactions with natural binding partners. Also disclosed are methods for diagnosing and treating specific abnormal conditions in an organism with mCLK2, mCLK3, and mCLK4 related molecules or compounds. The nucleic acid molecules, nucleic acid vectors, recombinant cells, polypeptides, and antibodies may be produced using well known and standard techniques used currently in the art.
Thus in a first aspect, the invention features isolated, enriched, or purified nucleic acid molecules encoding a novel mCLK2, mCLK3, or mCLK4 polypeptide.
The term xe2x80x9cisolatedxe2x80x9d, in reference to nucleic acid molecules, indicates that a naturally occurring sequence has been removed from its normal cellular environment. The isolated nucleic acid of the present invention is unique in the sense that it is not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular (i.e., chromosomal) environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not imply that the sequence is the only nucleotide chain present, but that it is essentially free (about 90-95% pure at least) of non-nucleotide material naturally associated with it, and thus is distinguished from isolated chromosomes.
The term xe2x80x9cenrichedxe2x80x9d, in reference to nucleic acid molecules, means that the specific DNA or RNA sequence constitutes a significantly higher fraction (2-5 fold) of the total DNA or RNA present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was taken. A person skilled in the art could enrich a nucleic acid mixture by preferentially reducing the amount of other DNA or RNA present, or preferentially increasing the amount of the specific DNA or RNA, or both. However, nucleic acid molecule enrichment does not imply that there is no other DNA or RNA present, the term only indicates that the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantlyxe2x80x9d qualifies xe2x80x9cincreasedxe2x80x9d to indicate that the level of increase is useful to the person performing the recombinant DNA technique, and generally means an increase relative to other nucleic acids of at least 2 fold, or more preferably at least 5 to 10 fold or more. The term also does not imply that there is no DNA or RNA from other sources. Other DNA may, for example, comprise DNA from a yeast or bacterial genome, or a cloning vector. In addition, levels of mRNA may be naturally increased relative to other species of mRNA when working with viral infection or tumor growth techniques. This term distinguishes from naturally occurring events, such as viral infection, or tumor type growths, in which the level of one mRNA may be naturally increased relative to other species of mRNA. That is, the term is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired nucleic acid.
It is also advantageous for some purposes that a nucleotide sequence be in purified form. The term xe2x80x9cpurifiedxe2x80x9d in reference to nucleic acid does not require absolute purity (such as a homogeneous preparation). Instead, it represents an indication that the sequence is relatively more pure than in the natural environment (compared to the natural level this level should be at least 2-5 fold greater, e.g., in terms of mg/mL). Individual clones isolated from a cDNA library may be purified to electrophoretic homogeneity. The claimed DNA molecules obtained from these clones could be obtained directly from total DNA or from total RNA. The cDNA clones are not naturally occurring, but rather are preferably obtained via manipulation of a partially purified naturally occurring substance (messenger RNA). The construction of a cDNA library from mRNA involves the creation of a synthetic substance (cDNA) and pure individual cDNA clones can be isolated from the synthetic library by clonal selection of the cells carrying the cDNA library. Thus, the process, which includes the construction of a cDNA library from mRNA and isolation of distinct cDNA clones, yields an approximately 106-fold purification of the native message. Thus, purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is expressly contemplated.
The term xe2x80x9cnucleic acid moleculexe2x80x9d describes a polymer of deoxyribonucleotides (DNA) or ribonucleotides (RNA) The nucleic acid molecule may be isolated from a natural source by cDNA cloning or subtractive hybridization or synthesized manually. The nucleic acid molecule may be synthesized manually by the triester synthetic method or by using an automated DNA synthesizer.
xe2x80x9ccDNA cloningxe2x80x9d techniques refer to hybridizing a small nucleic acid molecule, a probe, to genomic cDNA that is bound to a membrane. The probe hybridizes (binds) to complementary sequences of cDNA. The term xe2x80x9ccomplementaryxe2x80x9d describes two nucleotides that can form multiple favorable interactions with one another. For example, adenine is complementary to thymidine as they can form two hydrogen bonds. Similarly, guanine and cytosine are complementary since they can form three hydrogen bonds. cDNAs are molecules that are reverse transcribed from fragments of message RNA from a genomic source. These fragments form a cDNA library of nucleic acid molecules. cDNA libraries are constructed from natural sources such as mammalian blood, semen, or tissue.
The term xe2x80x9csubtractive hybridizationxe2x80x9d refers to a method similar to cDNA-cloning except that cDNA prepared from mRNA in unstimulated cells is added to mRNA in stimulated or different types of cells. cDNA/mRNA can then be precipitated to enrich the mRNA specific to the stimulation signal or different cell.
The term xe2x80x9chybridizexe2x80x9d refers to a method of interacting a nucleic acid probe with a DNA or RNA molecule in solution or on a solid support, such as cellulose or nitrocellulose. If a nucleic acid probe binds to the DNA or RNA molecule with high affinity, it is said to xe2x80x9chybridizexe2x80x9d to the DNA or RNA molecule. As mentioned above, the strength of the interaction between the probe and its target can be assessed by varying the stringency of the hybridization conditions. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acids having one or two mismatches out of 20 contiguous nucleotides.
By xe2x80x9cnovelxe2x80x9d is meant new and in the particular context of the present invention refers to CLK sequences that have not been previously described. In preferred embodiments the novel sequence may be the full-length serine CLK2 or CLK3 sequence, the full-length mammalian CLK4 sequence, or shorter fragments (preferably functional fragments) if any of the above as long as they were not already previously described.
The terms xe2x80x9cmCLK2xe2x80x9d, xe2x80x9cmCLK3xe2x80x9d, and xe2x80x9cmCLK4xe2x80x9d refer to polypeptides that have amino acid sequences substantially similar to those set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6. A sequence that is substantially similar will preferably have at least 95% identity, more preferably at least 96-97% identity, and most preferably 98-100% identity to the sequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6. CLK protein kinase polypeptides preferably have protein kinase activity and fragments of the full length CLK protein kinase sequences having such activity may be identified using techniques well known in the art, such as sequence comparisons and assays such as those described in the examples herein. Other aspects of mCLK2, mCLK3, and mCLK4 nucleic acid sequences, amino acid sequences, functions and properties are further depicted in Nayler et al., 1997, Biochem J. 326: 693-700, hereby incorporated by reference herein in its entirety including all figures, tables, and drawings.
By xe2x80x9cidentityxe2x80x9d is meant a property of sequences that measures their similarity or relationship. Generally speaking, identity is measured by dividing the number of identical residues by the total number of residues and gaps and multiplying the product by 100. xe2x80x9cGapsxe2x80x9d are spaces in an alignment that are the result of additions or deletions of amino acids. Thus, two copies of exactly the same sequence have 100% identity, but sequences that are less highly conserved, and have deletions, additions, or replacements, may have a lower degree of identity. Those skilled in the art will recognize that several computer programs are available for determining sequence identity.
A preferred embodiment of the invention concerns nucleic acid molecules relating to mCLK2, mCLK3, and mCLK4 that are enriched, isolated, or purified from a mammalian source. These nucleic acid molecules can be isolated from, among others, blood, semen, or tissue. Although mCLK2, mCLK3, and mCLK4 nucleic acid molecules are isolated from mouse cells, current recombinant DNA techniques can readily elucidate related nucleic acid molecules in other mammalian tissue. Mammals include, but are not limited to, mice, rats, rabbits, cows, horses, monkeys, apes, and preferably humans.
Another preferred embodiment of the invention concerns isolated nucleic acid molecules that encode at least seventeen amino acids of a mCLK2, mCLK3, or mCLK4 polypeptide. Preferably, at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 450, 475, or 485 contiguous amino acids are encoded. This preferred embodiment of the invention is achieved by applying routine recombinant DNA techniques known to those skilled in the art.
Another aspect of the invention features a nucleic acid probe that can detect nucleic acid molecules encoding a mCLK2, mCLK3, or mCLK4 polypeptide in a sample.
The term xe2x80x9cnucleic-acid probexe2x80x9d refers to a nucleic acid molecule that is complementary to and can bind a nucleic acid sequence encoding the amino acid sequence substantially similar to that set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6.
The nucleic acid probe or its complement encodes any one of the amino acid molecules set forth in the invention. Thus the nucleic acid probe can encode at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 450, 475, or 485 contiguous amino acids of the full-length sequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6.
The nucleic acid probe can be labeled with a reporter molecule or molecules. The term xe2x80x9creporter moleculexe2x80x9d refers to a molecule that is conjugated to the nucleic acid probe or is contained within the nucleic acid probe. The reporter molecule allows the detection of the probe by methods used in the art. Reporter molecules are chosen from, but not limited to, the group consisting of an enzyme, such as a peroxidase, a radioactive element, or an avidin or biotin molecule.
A nucleic acid probe, whether labeled or unlabeled, should hybridize to a complement in a sample. Various low or high stringency hybridization conditions may be used depending upon the specificity and selectivity desired. Stringency is controlled by varying salt or denaturant concentrations. Under stringent hybridization conditions only highly complementary nucleic acid sequences hybridize. Preferably, such conditions prevent hybridization of nucleic acid molecules having one or two mismatches out of 20 contiguous nucleotides, more preferably prevent hybridization of nucleic acid molecules having one mismatch out of 35 contiguous nucleotides, and most preferably prevent hybridization of nucleic acid molecules having one mismatch out of 50 contiguous nucleotides.
The nucleic acid probe or complement can also refer to a nucleic acid molecule encoding a conserved or unique region of amino acids. These nucleic acid molecules are useful as hybridization probes to identify and clone additional polypeptides relating to CLK serine/threonine kinases. The term xe2x80x9cconserved nucleic acid regionsxe2x80x9d refers to regions present in two or more nucleic acid molecules encoding a CLK protein kinase polypeptide, to which a particular nucleic acid sequence can hybridize under lower stringency conditions. Examples of lower stringency conditions suitable for screening nucleic acid molecules are provided in Abe, et al. J. Biol. Chem., 19:13361 (1992) (hereby incorporated by reference herein in its entirety, including any drawings). Preferably, conserved regions differ by no more than 5 out of 20 nucleotides, more preferably conserved regions differ by no more than 10 out of 20 nucleotides, and most preferably conserved regions differ by no more than 15 out of 20 nucleotides. Protein kinases share conserved regions in the catalytic domain.
The term xe2x80x9cunique nucleic acid regionxe2x80x9d concerns a sequence present in a full length nucleic acid molecule encoding a CLK protein kinase polypeptide that is not present in a sequence encoding any other naturally occurring polypeptide. Such regions preferably comprise 30 or 45 contiguous nucleotides, more preferably 100 contiguous nucleotides, and most preferably 200 contiguous nucleotides present in the full length nucleic acid sequence encoding a CLK protein kinase polypeptide. In particular, a unique nucleic acid region is preferably of mammalian origin.
Methods for using the probes include detecting the presence or amount of CLK protein kinase RNA in a sample by contacting the sample with a nucleic acid probe under conditions such that hybridization occurs and detecting the presence or amount of the probe bound to CLK RNA. The nucleic acid duplex formed between the probe and a nucleic acid sequence encoding a CLK protein kinase polypeptide may be used in the identification of the sequence of the nucleic acid detected (for example see, Nelson et al., in Nonisotopic DNA Probe Techniques, p. 275 Academic Press, San Diego (Kricka, ed., 1992) hereby incorporated by reference herein in its entirety, including any drawings). Kits for performing such methods may be constructed to include a container holding a nucleic acid probe.
In yet another aspect, the invention relates to a nucleic acid vector comprising a promoter element and a nucleic acid molecule described in the first aspect of the invention.
The term xe2x80x9cnucleic acid vectorxe2x80x9d relates to a single or double stranded circular nucleic acid molecule that can be transfected or transformed into cells and replicate independently or within a cell genome. A vector can be cut and thereby linearized upon treatment with restriction enzymes. An assortment of vectors, restriction enzymes, and the knowledge of the nucleotide sequences that the restriction enzymes operate upon are readily available to those skilled in the art. A nucleic acid molecule encoding a CLK protein kinase can be inserted into a vector by cutting the vector with restriction enzymes and ligating the two pieces together.
The term xe2x80x9cpromoter elementxe2x80x9d describes a nucleotide sequence that is incorporated into a vector that, once inside an appropriate cell, may facilitate transcription factor and/or polymerase binding and subsequent transcription of portions of the vector DNA into mRNA. The promoter element precedes the SI end of the nucleic acid molecule of the first aspect of the invention such that the latter is transcribed into mRNA. Recombinant cell machinery then translates mRNA into a polypeptide.
Many techniques are available to those skilled in the art to facilitate transformation or transfection of the nucleic acid vector into a prokaryotic or eukaryotic organism. The terms xe2x80x9ctransformationxe2x80x9d and xe2x80x9ctransfectionxe2x80x9d refer to methods of inserting a nucleic acid vector into a cellular organism. These methods involve a variety of techniques, such as treating the cells with high concentrations of salt, an electric field, or detergent, to render the cell outer membrane or wall permeable to nucleic acid molecules of interest.
A nucleic acid vector can be useful for identifying natural binding partners of CLK serine/threonine kinases.
The term xe2x80x9cnatural binding partnersxe2x80x9d refers to polypeptides that bind to CLK serine/threonine kinases and play a role in propagating a signal in a signal transduction process. The term xe2x80x9cnatural binding partnerxe2x80x9d also refers to a polypeptide that binds to CLK serine/threonine kinases within a cellular environment with high affinity. High affinity represents an equilibrium binding constant on the order of 10-1 M. However, a natural binding partner can also transiently interact with a CLK protein kinase and chemically modify it. CLK protein kinase natural binding partners are chosen from a group consisting of, but not limited to, src homology 2 (SH2) or 3 (SH3) domains, other phosphoryl tyrosine binding domains, and receptor and non-receptor protein kinases or protein, phosphatases.
Methods are readily available in the art for identifying binding partners of polypeptides of interest. These methods include screening cDNA libraries included in one nucleic acid vector with a nucleic acid molecule encoding the desired polypeptide in another nucleic acid vector. Vojtek et al., 1993, Cell 74:205-214. These techniques often utilize yeast recombinant cells. These techniques also utilize two halves of a transcription factor, one half that is fused to a polypeptide encoded by the cDNA library and the other that is fused to the polypeptide of interest. Interactions between a polypeptide encoded by the cDNA library and the polypeptide of interest are detected when their interaction concomitantly brings together the two halves into an active transcription factor which in turn activates a gene that reports the interaction. Any of the nucleic molecules encoding mCLK2, mCLK3, or mCLK4 can be readily incorporated into an nucleic acid vector used in such a screening procedure by utilizing standard recombinant DNA techniques in the art.
Another aspect of the invention relates to a recombinant cell or tissue comprising a nucleic acid molecule encoding a mCLK2, mCLK3, or mCLK4 polypeptide.
The term xe2x80x9crecombinantxe2x80x9d refers to an organism that has a new combination of genes or nucleic acid molecules. A new combination of genes or nucleic acid molecules can be introduced to an organism using a wide array of nucleic acid manipulation techniques available to those skilled in the art.
The recombinant cell can be a eukaryotic or prokaryotic organism. The term xe2x80x9ceukaryotexe2x80x9d refers to an organism comprised of cells containing a nucleus. Eukaryotes are differentiated from xe2x80x9cprokaryotesxe2x80x9d which do not house their genomic DNA inside a nucleus. Prokaryotes include unicellular organisms such as bacteria while eukaryotes are represented by yeast, invertebrates, and vertebrates.
The recombinant cell can also harbor a nucleic acid vector that is extragenomic. The term xe2x80x9cextragenomicxe2x80x9d refers to a nucleic acid vector which does not integrate into a cell genome. Many nucleic acid vectors are designed with their own origins of replication which allow them to utilize the recombinant cell replication machinery to copy and propagate the nucleic acid vector nucleic acid sequence. These nucleic acid vectors are small enough that they are not likely to harbor nucleic acid sequences homologous to genomic sequences of the recombinant cell. Thus these nucleic acid vectors replicate independently of the genome and do not recombine with or integrate into the genome.
A recombinant cell can also harbor a portion of a nucleic acid vector in an intragenomic fashion. The term xe2x80x9cintragenomicxe2x80x9d defines a nucleic acid vector that integrates within a cell genome. Multiple nucleic acid vectors available to those skilled in the art contain nucleic acid sequences that are homologous to nucleic acid sequences in a particular organism""s genomic DNA. These homologous sequences will result in recombination events that incorporate portions of the nucleic acid vector into the genomic DNA. Those skilled in the art can control which nucleic acid sequences of the nucleic acid vector integrate into the cell genome by flanking the portion to be integrated into the genome with homologous sequences in the nucleic acid vector.
In yet another aspect, the invention features an isolated, enriched, or purified polypeptide encoded by a mCLK2, mCLK3, or mCLK4 nucleic acid molecule of the invention.
The term xe2x80x9cisolatedxe2x80x9d, in reference to a polypeptide, describes a polymer of amino acids conjugated to each other that are separated from a natural source. The polypeptide can also be synthesized manually. Isolated peptides can be at least 17, 20, 25, 30, 35, 40, 50, 100, 200, or 300 contiguous amino acids of one of the full-length sequences set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6. In certain aspects longer polypeptides are preferred, such as those with 400, 450, 475, or 485 of the contiguous amino acids of mCLK2, mCLK3, or mCLK4 set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6.
The isolated polypeptides of the present invention are unique in the sense that they are not found in a pure or separated state in nature. Use of the term xe2x80x9cisolatedxe2x80x9d indicates that a naturally occurring sequence has been removed from its normal cellular environment. Thus, the sequence may be in a cell-free solution or placed in a different cellular environment. The term does not-imply that the sequence is the only amino acid chain present, but that it is essentially free (about 90-95% pure at least) of non-amino aced material naturally associated with it.
The term xe2x80x9cenrichedxe2x80x9d, in reference to a polypeptide, defines a specific amino acid sequence constituting a significantly higher fraction (2-5 fold) of the total of amino acids present in the cells or solution of interest than in normal or diseased cells or in the cells from which the sequence was separated. A person skilled in the art can preferentially reduce the amount of other amino acid sequences present, or preferentially increase the amount of specific amino acid sequences of interest, or both. However, the term xe2x80x9cenrichedxe2x80x9d does not imply that there are no other amino acid sequences present. Enriched simply means the relative amount of the sequence of interest has been significantly increased. The term xe2x80x9csignificantxe2x80x9d indicates that the level of increase is useful to the person making such an increase. The term also means an increase relative to other amino acids of at least 2 fold, or more preferably at least 5 to 10 fold, or even more. The term also does not imply that there are no amino acid sequences from other sources. Other source amino acid sequences may, for example, comprise amino acid sequences from a recombinant organism. xe2x80x9cEnrichedxe2x80x9d is meant to cover only those situations in which a person has intervened to elevate the proportion of the desired amino acid sequence.
The term xe2x80x9cpurifiedxe2x80x9d, in reference to a polypeptide, does not require absolute purity (such as a homogeneous preparation); instead, it represents an indication that the amino acid sequence is relatively more pure than in a cellular environment. The concentration of the preferred amino acid sequence should be at least 2-5 fold greater (in terms of mg/ml) than its concentration in a cellular environment. Purification of at least one order of magnitude, preferably two or three orders, and more preferably four or five orders of magnitude is preferred. The substance is preferably free of contamination, as indicated by purity levels of 90%, 95%, or 99%.
A preferred embodiment of the invention relates to a mCLK2, mCLK3, or mCLK4 polypeptide that is a unique fragment. This unique fragment can contain at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 450, 475, or 485 contiguous amino acids of one of the full-length sequences. In addition, preferred lengths and portions of mCLK2, mCLK3, or mCLK4 amino acid sequences are encoded by the nucleic acid molecules defined in the first aspect of the invention.
The term xe2x80x9cunique fragmentxe2x80x9d refers to the minimum stretch of amino acids in one mCLK molecule that is different in sequence than any other portion of another protein kinase. Since the largest identical stretch of amino acids found in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 is seventeen amino acids, the minimum unique fragment for a mCLK protein kinase is seventeen amino acids.
The polypeptide can be isolated, enriched, or purified from a prokaryotic or eukaryotic recombinant cell. A eukaryotic cell includes mammals and preferably humans. Multiple standard techniques are available to those skilled in the art to facilitate isolation, enrichment, or purification of a polypeptide from recombinant cells. These methods typically include lysing the recombinant cells and separating the polypeptide of interest from the rest of the cell polypeptides, nucleic acids, and fatty acid-based material using standard chromatography techniques known in the art.
Another aspect of the invention features an antibody, that is monoclonal or polyclonal, or an antibody fragment having specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide.
Antibodies or antibody fragments are polypeptides with regions that can bind to other polypeptides with high affinity. The term xe2x80x9cspecific binding affinityxe2x80x9d describes an antibody that binds to a mCLK2, mCLK3, or mCLK4 polypeptide with greater affinity than it binds to other polypeptides under specified conditions.
The term xe2x80x9cpolyclonalxe2x80x9d refers to a mixture of antibodies with specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide, while the term xe2x80x9cmonoclonalxe2x80x9d refers to one antibody with specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide. A monoclonal antibody binds to one specific region on a mCLK2, mCLK3, or mCLK4 polypeptide and a polyclonal mixture of antibodies can bind multiple regions of a mCLK2, mCLK3, or mCLK4 polypeptide. One skilled in the art would note that a monoclonal and especially a polyclonal antibody that has specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide will most likely also have specific binding affinity to another CLK protein kinase polypeptide of mammalian origin.
The term xe2x80x9cantibody fragmentxe2x80x9d refers to a portion of an antibody, often the hypervariable region and portions of the surrounding heavy and light chains, that displays specific binding affinity for a particular molecule. A hypervariable region is a portion of an antibody that physically binds to the ligand to which it binds specifically.
Antibodies or antibody fragments having specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide may be used in methods for detecting the presence and/or amount of a CLK protein kinase polypeptide in a sample by probing the sample with the antibody under conditions suitable for CLK protein kinase-antibody immunocomplex formation and detecting the presence and/or amount of the antibody conjugated to a CLK protein kinase polypeptide. Diagnostic kits for performing such methods may be constructed to include antibodies or antibody fragments specific for a CLK protein kinase as well as a conjugate of a binding partner of the antibodies or the antibodies themselves.
Another aspect of the invention features a hybridoma which produces an antibody having specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide. A xe2x80x9chybridomaxe2x80x9d is an immortalized cell line which is capable of secreting an antibody, for example an antibody with specific binding affinity to a mCLK2, mCLK3, or mCLK4 polypeptide.
Another aspect of the invention relates to an isolated, enriched, or purified nucleic acid molecule comprising a nucleotide sequence that: (a) encodes a full length mCLK2, mCLK3, or mCLK4 amino acid sequence as set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6; (b) encodes the complement of the nucleotide sequence encoding the amino acid sequences of FIG. 1, FIG. 2, FIG. 4, or FIG. 6; (c) hybridizes under highly stringent conditions to the nucleic acid molecule of (a) and encodes a naturally occurring mCLK2, mCLK3, or mCLK4 protein; (d) a mCLK2, mCLK3, or mCLK4 protein having the full length amino acid sequence as set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 except that it lacks one or more of the following segments of amino acid residues 1-182, 183-470, or 471499 of mCLK2, 1-176, 177-473, or 474-496 of mCLK3, or 1183, 184-486, or 486-489 of mCLK4; (e) the complement of the nucleotide sequence of (d); (f) a polypeptide having the amino acid sequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 from amino acid residues 1-182, 183-470, or 471-499 of mCLK2, 1176-, 177473, or 474-496 of mCLK3, or 1-183, 184-486, or 486-489 of mCLK4;(g) the complement of the nucleotide sequence of (f); (h) encodes a polypeptide having the full length amino acid sequence set forth in FIG. 1, FIG. 2, FIG. 4, or FIG. 6 except that it lacks one or more of the domains selected from the group consisting of a N-terminal domain, a catalytic domain, and a C-terminal-region; or (i) the complement of the nucleotide sequence of (h).
The term xe2x80x9cN-terminal domainxe2x80x9d refers to a portion of the full length mCLK2, mCLK3, or mCLK4 amino acid sequences spanning from the amino terminus to the start of the catalytic domain.
The term xe2x80x9ccatalytic domainxe2x80x9d refers to a portion of the full length mCLK2, mCLK3, or mCLK4 amino acid molecules that does not contain the N-terminal domain or C-terminal region and has catalytic activity.
The term xe2x80x9cC-terminal regionxe2x80x9d refers to a portion of the full length mCLK2, mCLK3, or mCLK4 amino acid molecules that begins at the end of the catalytic domain and ends at the carboxy terminal amino acid, which is the last amino acid encoded before the stop codon in the nucleic acid sequence.
Domains are regions of polypeptides which contain particular functions. For instance, N-terminal or C-terminal domains of signal transduction proteins can serve functions including, but not limited to, binding molecules that localize the signal transduction molecule to different regions of the cell or binding other signaling molecules directly responsible for propagating a particular cellular signal. Some domains can be expressed separately from the rest of the protein and function by themselves, while others must remain part of the intact protein to retain function. The latter are termed functional regions of proteins and also relate to domains.
Functional regions of mCLK2, mCLK3, or mCLK4 may be identified by aligning their amino acid sequences with amino acid sequences of other polypeptides with known functional regions. If regions of mCLK2, mCLK3, or mCLK4 share high amino acid identity with the amino acid sequences of known functional regions, then mCLK2, mCLK3, or mCLK4 can be determined to contain these functional regions by those skilled in the art. The functional regions can be determined, for example, by using computer programs and sequence information available to those skilled in the art.
Other functional regions of signal transduction molecules that may exist within mCLK2, mCLK3, or mCLK4 include, but are not limited to, proline-rich regions or phosphoryl tyrosine regions. These regions can interact with natural binding partners such as SH2 or SH3 domains of other signal transduction molecules. Another aspect of the invention relates to nucleic acid vectors comprising any of the nucleic acid molecules described herein.
In another aspect, the invention includes recombinant cells or tissues comprising any of the nucleic acid molecules described herein.
In yet another aspect, the invention relates to a method of identifying compounds capable of inhibiting or activating CLK protein kinase phosphorylation activity. This method comprises the following steps: (a) adding a compound to a mixture comprising a CLK protein kinase polypeptide and a substrate for a CLK protein kinase; and (b) detecting a change in phosphorylation of said substrate.
The term xe2x80x9ccompoundxe2x80x9d includes small organic molecules including, but not limited to, oxindolinones, quinazolines, tyrphostins, quinoxalines, and extracts from natural sources.
The term xe2x80x9cCLK protein kinase polypeptidexe2x80x9d refers to any CLK protein kinase isolated from an insect or a mammal. The polypeptide can be the full length amino acid sequence (the contiguous amino acids encoded by the nucleic acids spanning from the start codon to the stop codon of a naturally occurring CLK protein kinase nucleic acid molecule) or portions of a naturally occurring full length CLK protein kinase. Preferably, at least 17, 20, 25, 30, 35, 40, 50, 100, 200, 300, 400, 450, 475, or 485 contiguous amino acids are encoded for the CLK protein kinase polypeptide.
The term xe2x80x9ca change in phosphorylationxe2x80x9d, in the context of the invention, defines a method of observing a change in phosphorylation of the substrate in response to adding a compound to cells. The phosphorylation can be detected, for example, by measuring the amount of a substrate that is converted to a product with respect to time. Addition of a compound to cells expressing a CLK protein kinase polypeptide may either enhance (activate) or lower (inhibit) the phosphorylation. If a compound lowers phosphorylation, the compound is assumed to bind to a CLK protein kinase polypeptide and block the ability of CLK protein kinase to bind and/or turn over a substrate. If a compound enhances phosphorylation, the compound is assumed to bind to a CLK protein kinase polypeptide and facilitate the ability of CLK protein kinase to bind and/or turn over a substrate.
The method can utilize any of the molecules disclosed in the invention. These molecules include nucleic acid molecules encoding mCLK2, mCLK3, or mCLK4 polypeptides, nucleic acid vectors, recombinant cells, polypeptides, or antibodies of the invention.
Another aspect of the invention is a method of identifying compounds useful for the diagnosis or treatment of an abnormal condition in an organism. The abnormal condition can be associated with an aberration in a signal transduction pathway characterized by an interaction between a CLK protein kinase polypeptide and a natural binding partner. The method comprises the following steps: (a) adding a compound to cells; and (b) detecting whether the compound promotes or disrupts said interaction between a CLK protein kinase polypeptide and a natural binding partner.
The term xe2x80x9cabnormal conditionxe2x80x9d refers to a function in an organism""s cells or tissue that deviate from a normal function in the cells or tissue of that organism. In the context of this aspect of the invention, abnormal conditions can be associated with cell proliferation or with RNA splicing.
Aberrant cell proliferative conditions include cancers such as fibrotic and mesangial disorders, abnormal angiogenesis and vasculogenesis, wound healing, psoriasis, diabetes mellitus, and inflammation.
RNA splicing is a necessary function of a cell that occurs in a cell nucleus. This process is the last step in the synthesis of message RNA from DNA. One molecule of RNA transcribed from DNA is tied into a lariat, incised in at least two places at the intersection of the strands, the lariat is excised, and the non-excised portion is ligated together. The modified RNA is then fit to be message RNA and is ejected from the cell nucleus to be translated into a polypeptide. Thus any aberrations that exist in an organisms ability to splice the RNA of a particular gene could result in the deficiency of a cellular agent and give rise to an abnormal condition.
Thus, regulating RNA splicing could be useful in treating cancer. For example, it is known that proteins such as Raf or src become oncogenic when made in a truncated form, such as could happen when RNA is incorrectly spliced. For this reason, the proteins of the invention might be useful for finding compounds to treat cancer. In addition, molecules involved in RNA processing have been linked to spermatogenesis. Thus, modifying RNA processing could lead to more sperm (to treat infertility) or less sperm. These methods would preferably involve CLK3 due to its high expression in the testis.
The abnormal condition can be diagnosed when the organism""s cells exist within the organism or outside of the organism. Cells existing outside the organism can be maintained or grown in cell culture dishes. For cells harbored within the organism, many techniques exist in the art to administer compounds, including (but not limited to) oral, parenteral, dermal, and injection applications. For cells outside of the patient, multiple techniques exist in the art to administer the compounds, including (but not limited to) cell microinjection techniques, transformation techniques, and carrier techniques.
The term xe2x80x9csignal transduction pathwayxe2x80x9d refers to the molecules that propagate an extracellular signal through the cell membrane to become an intracellular signal. This signal can then stimulate a cellular response. The polypeptide molecules involved in signal transduction processes are typically receptor and non-receptor protein kinases, receptor and non-receptor protein phosphatases, and transcription factors.
The term xe2x80x9caberrationxe2x80x9d, in conjunction with a signal transduction process, refers to a CLK protein kinase polypeptide that is over- or under-expressed in an organism, mutated such that its catalytic activity is lower or higher than wild-type CLK protein kinase, mutated such that it can no longer interact with a binding partner, is no longer modified by another protein kinase or protein phosphatase, or no longer interacts with a binding partner.
The term xe2x80x9cinteractionxe2x80x9d defines the complex formed between a CLK protein kinase polypeptide and a natural binding partner. Compounds can bind to either the CLK protein kinase polypeptide or the natural binding partner and disrupt the interaction between the two molecules. The method can also be performed by administering a group of cells containing an aberration in a signal transduction process to an organism and monitoring the effect of administering a compound on organism function. The art contains multiple methods of introducing a group of cells to an organism as well as methods of administering a compound to an organism. The organism is preferably an animal such as a frog, mouse, rat, rabbit, monkey, or ape, and also a human.
Methods of determining a compound""s effect of detecting an interaction between CLK serine/threonine kinases and natural binding partners exist in the art. These methods include, but are not limited to, determining the effect of the compound upon the catalytic activity of a CLK protein kinase polypeptide, the phosphorylation state of the CLK protein kinase polypeptides or natural binding partners, the ability of a CLK protein kinase to bind a natural binding partner, or a difference in a cell morphology.
Differences in cell morphology include growth rates, differentiation rates, cell hypertrophy, survival, or prevention of cell death. These phenomena are-simply measured by methods in the art. These methods can involve observing the number of cells or the appearance of cells under a microscope with respect to time (days).
Another aspect of the invention relates to a method of diagnosing an abnormal condition associated with cell proliferation or RNA splicing in an organism. The abnormal condition can be associated with an aberration in a signal transduction pathway characterized by an interaction between a CLK protein kinase polypeptide and a natural binding partner. The method comprises the step of detecting the abnormal interaction.
The abnormal interaction can be assessed by the methods described above in reference to the identification of compounds useful for diagnosing an abnormal condition in an organism.
In a final aspect, the invention features a method of administering a compound to a male organism that acts a contraceptive to reproduction. The compound can inhibit the catalytic activity of a CLK protein kinase or inhibit the binding of a natural binding partner to a CLK protein kinase.
Preferred embodiments of the methods of the invention relate to CLK serine/threonine kinases that are isolated from mammals, preferably humans, and to organisms that are mammals, preferably humans.
The summary of the invention described above is not limiting and other features and advantages of the invention will be apparent from the following detailed description of the invention, and from the claims.